KR101590666B1 - Irradiated membrane for cell expansion - Google Patents

Abstract

The present invention relates to a membrane which can be used for culturing adherent or floating cells, in particular adherent cells, wherein said membrane is in contact with a wet and dry membrane by gamma or beta radiation or electron beam at a dose of 12.5 kGy to 175 kGy in the presence of oxygen This study considers cell adhesion and proliferation. The resulting membrane may be used without any pretreatment by the surface modifying material. The present invention also relates to a method for producing the irradiated membrane, which can be used for culturing cells, especially adherent cells, and a method of using such membranes for culturing cells, especially adherent cells.

Description

[0001] IRRADIATED MEMBRANE FOR CELL EXPANSION FOR CELL PROLIFERATION [0002]

The present invention relates to a membrane which can be used for culturing adherent or suspension cells, in particular cohesive cells, wherein said membrane is exposed to a radioactivity in the presence of oxygen at a dose of 12.5-175 kGy by gamma or beta radiation or electron beam Considering the adhesion and proliferation of cells due to irradiation of wet or dry membranes. The resulting membrane may be used without any pretreatment by the surface modifying material. The present invention also relates to a method for producing said irradiated membrane which can be used for culturing cells, especially adherent cells, and to a method of using such membranes for culturing cells, particularly adherent cells.

It is an object of the present invention to identify membranes exhibiting growth characteristics substantially similar to tissue culture polystyrene (TCPS) plates, which present a current gold standard for cell proliferation using culture flasks or cell stacks. The main features to be measured are post-expansion features including cell growth rate, cell re-attachment efficiency on the membrane, and morphological control, phenotype and differentiation potential. Such membranes should be suitable for being produced in a variety of geometries, such as flat sheet membranes or hollow fiber membranes.

The present invention relates specifically to membranes that can be used, for example, in culturing, growing, storing and / or propagating various types of cohesive cells. In the context of the present invention, the expression "cell culturing" or "culturing of cells (culturing of cells)" (cultivation of cells) is intended to encompass all such uses, i.e., adhesion, retention, growth, proliferation, differentiation, , Transfection) and / or storage.

Like most cells in vivo, many cells are adherent cells or anchorage dependent cells. That is, the cells can metabolize or divide only as long as they are attached to the surface or substrate. Cells of the circulatory system (e.g., lymphocytes and red blood cells) and other cell types such as hematopoietic stem cells, hepatocytes, CHO cells, etc., grow in an unattached and floating state in solution in vitro. While many adherence-dependent cells can grow on free or synthetic surfaces, these cells often lose their ability to differentiate and respond to hormones. Loss of cell morphology not only necessitates loss of function, but also interferes with regeneration in long-term culture systems. However, it is very important to use human cells for long-term culture, for example tissue culture, and many cells are not available in any quantity. For this reason, such tissue culture dishes are often coated with extracellular matrix components, such as collagen or fibronectin. However, the use of xenogenic factors, particularly when cells on the cell itself or on the substrate are used for medical treatment in humans, is a distinct disadvantage, which leads to a risk of contamination and consequently to adverse reactions in the patient under treatment It is because.

The failure of a cell to grow on its surface or maintain its ability is a major limitation of current tissue culture techniques, for example. Tissue culture fluids are potential sources of tissues and organs that can be used for transplantation into humans. For example, tissue cultured skin cells can potentially be used in skin grafts. This objective may be achieved, for example, by the use of a non-cellular substrate that is dependent on the ability of the body to regenerate for proper orientation and orientation of new tissue growth, or by the use of a cell- and is intended to develop biological substituents can be maintained (Atala (2006):.. .. Recent developments in tissue engineering and regenerative medicine Curr Opin Pediatr 16, 167-171). Cells can also be used for therapy via infusion with carriers or alone. In this case, the cells need to be propagated in the culture medium, attached to the support substrate, and then re-implanted into the host after proliferation. Currently veterinary therapeutic applications are available and may represent additional uses for membranes in cell culture.

  The ability to cultivate cells, particularly adherent cells, is also important because the cells represent biological "factories" capable of producing large amounts of bioproducts such as growth factors, antibodies and viruses.

In addition, cell culture has emerged as a tool for biocompatibility and toxicology research in the pharmaceutical and life sciences industries.

Finally, tissue culture generally includes cells derived from only one or a few tissues or organs. As a result, cell culture provides scientists with a system for studying the characteristics of individual cell types without the complexity of studying using whole organisms.

Known methods for culturing adherent cells include hollow fiber membrane bioreactors. In this system, the cells are generally attached to the lumen of a cylindrical hollow fiber membrane. The culture medium and oxygen flow through the center of the cylindrical hollow fiber membrane. The molecular weight cut-off of the membrane allows the nutrients and oxygen to reach the cells without allowing the cells to exit.

A variety of polymers have been proposed for making semipermeable membranes for cell and tissue culture (US 2007/269489). The polymer may be selected from the group consisting of polylaurates, polyvinyl chloride, polyvinylidene fluoride, polyurethane isocyanate, cellulose acetate, cellulose diacetate, cellulose triacetate, cellulose nitrate, polysulfone, polyethersulfone, polystyrene, polyurethane, poly Vinyl alcohol, polyacrylonitrile, polyamide, polymethylmethacrylate, polytetrafluoroethylene, polyethylene oxide, and combinations of these polymers. The polymeric support may also be composed of polyethylene terephthalate (PET) or polycarbonate. For example, additional materials that have been proposed as scaffolds for implantable tissue materials include celluloses or macroporous collagen carriers or biodegradable substrates.

WO 93/00439 A1 describes a process for maintaining a cell culture medium in a biocompatible semipermeable membrane in which cells are stimulated to secrete an activating factor. The semipermeable membrane used allows the diffusion of the active agent through the membrane while excluding the harmful drug present in the external environment from obtaining access to the culture medium. The membranes described have a tubular shape and are said to enable the diffusion of molecules with molecular weights below 150 kDa. The materials proposed for the membrane are acrylic copolymers, polyvinyl chloride, polystyrene, polyurethane, polyamide, polymethacrylate, polysulfone, polyacrylate, polyvinylidene fluoride, polyurethane-isocyanate, polyallylate , Cellulose acetate, polysulfone, polyvinyl alcohol, polyacrylonitrile, polyethylene oxide, and derivatives and mixtures thereof. The use of any irradiation to produce such membranes is not described. In the context of the disclosure, the membrane should act as a substrate for cell adhesion, but in fact it is not.

WO 90/11820 A2 discloses flat membranes or in vivo artificial implants with surfaces that can be used for in vitro cell growth. The membrane is described as being porous with a pore size in the range of 0.1 to 100 microns and having a finger-like configuration within the intermediate layer. The membrane comprises a hydrophobic polymer and a hydrophilic polymer. Examples given for hydrophobic polymers include polysulfones, polyamides, polyethersulfones, polyesters, polycarbonates, preferably polyetherurethanes, and copolymers thereof. The reference does not disclose whether the membrane can be used to coalesce and culture cells. The use of any survey technique is not described.

Apart from the problem of identifying membrane compositions that can be used as substrates for incubation of cohesive cells, currently known membranes in the art are capable of sufficiently promoting and maintaining adhesion, proliferation, differentiation and prolonged shelf life without pretreatment of the membranes or substrates Or from the addition of exogenous factors such as, for example, fibronectin, laminin or collagen.

See, for example, Fissell (2006), Expert Rev. Med . Devices 3 (2) , 155] outline the achievements in the development of artificial kidneys based on adhesion of renal tubular cells to synthetic polysulfone hollow fiber membranes. In this case, the membrane should be coated with ProNectin-L ™ to promote cell attachment.

Both of US-A 6,150,164 and US-A 6,942,879 disclose a sophisticated approach to bio-artificial kidneys based on kidney cells, for example endothelial cells or so-called kidney stem cells, which are seeded into hollow fibers have. Hollow fiber membranes which are said to be useful are based on cellulose, polyacrylonitrile, polysulfone and other components or copolymers thereof. The inner and outer surfaces of the hollow fibers are precoated with a suitable extracellular matrix component (EMC), including Type I collagen, type IV collagen, laminin, matrigel, proteoglycan, fibronectin and combinations thereof. Cells can be seeded only after such treatment.

Procedures for treating synthetic membranes, such as polysulfone-based membranes, with a gamma-irradiation process are known for cross-linking certain components of synthetic membranes, such as PVP, or for sterilizing synthetic membranes. The dose of radiation is generally in the range of 10 to 50 kGy, preferably in the range of 20 to 35 kGy (see, for example, US 6,960,297 B2 or US-A 6,103,117). Higher doses are avoided to minimize deterioration of the membrane. In addition, the known processes are generally designed for the same reason, omitting the presence of oxygen due to the formation of aggressive oxygen derivatives such as oxygen radicals and H ₂ O ₂ and membrane degradation by the oxygen derivatives. Sterilization by gamma-irradiation is generally carried out under aqueous conditions, i.e. using a wet membrane, where the solution is degassed to remove oxygen. When drying conditions are used, oxygen is also removed from the system by using an inert gas atmosphere, an oxygen scavenger, or the like.

In addition, the obvious purpose of the membrane and process of the prior art is to avoid or minimize adhesion of the cells to membranes commonly used in dialysis treatments. This contradicts the object of the present invention, which focuses on providing a desirable surface for cell adhesion and growth. Thus, the methods described in the prior art are designed to provide a membrane that is not useful for the purposes of the present invention, i. E., The cultivation of cells.

EP 1 795 254 A1 describes the formation of a polysulfone based membrane wherein the membrane has an oxygen concentration in the air around the membrane of from 0.001 to 0.1% and a moisture content of the membrane of from 0.2 to 7% by weight Under conditions, it is exposed to radiation, such as gamma rays. In this reference, it is mentioned that the use of a higher oxygen concentration, especially atmospheric air, results in an excited oxygen radical, destroying the main chain of the polymer and decomposing the polymer, and the atmosphere of an inert gas is preferably Has also been mentioned as preferred to have, in the presence of an oxygen scavenger. Since it is difficult to absolutely eliminate any oxygen, the above-mentioned limit value is presented as the highest oxygen value. It is further noted that EP 1 795 254 Al should have a radiation dose of 1 to 50 kGy, or more preferably 10 to 30 kGy, when using gamma radiation. Lower doses result in insufficient sterilization, while higher doses dissolve the components of the membrane. The reference does not consider using membranes for cell culture.

JP 2003/245526 also describes a method of irradiating hollow fiber membranes without the use of wet (water filled) fibers. In this method, the moisture content is adjusted to 4% or more based on the weight of the hollow fiber membrane. The oxygen concentration in the hollow fiber membrane module is set at 0.1 to 3.6%.

US 2006/191844 describes the treatment of a membrane module performed by filling with a degassed aqueous RO solution followed by sealing, where the module is then exposed to 10 to 60 kGy gamma radiation. When the dose of the gamma ray is too high, the hydrophobic polymer, the hydrophilic polymer and / or the housing may be degraded and deteriorated. Therefore, the dose of the gamma ray is preferably disclosed to be 50 kGy or less, particularly 30 kGy or less. When sloped water is used, dissolved oxygen in the water oxidizes and aggravates the components of the membrane.

WO 2006/135966 A1 discloses a method for crosslinking a hydrophilic component of a membrane, for example PVP, using gamma radiation, optionally in combination with a chemical solution treatment process. The dose used is 1 to 100 kGy, preferably 10 to 50 kGy. This irradiation is said to be applicable to dry or wet membranes. However, the examples presented use a wet membrane and a dose not exceeding 35 kGy. The use of membranes prepared according to the above references for cell culture is not mentioned.

EP 1 439 212 Al also describes the irradiation of polysulfone and PVP-based membranes with gamma radiation. Likewise, the membrane is irradiated in the wet state, where the membrane must contain at least 1 wt% water or be immersed in water and should not exceed 50 kGy to avoid any deterioration of the membrane. The irradiation reduces adhesion of the platelets to the membrane surface, which is in contrast to the object of the present invention, namely adhesion of the cells to the membrane surface.

JP 2004/305840 discloses a hollow fiber membrane composed of a hydrophilic polymer and a hydrophilic polymer which is sterilized by irradiation with a gamma ray after spinning. The irradiation is carried out in a dry, cold, deoxygenated, hermetically sealed state. It is also important to note that the exclusion of oxygen is crucial.

Summary of the Invention

The present invention discloses a membrane that is treated with beta rays or gamma rays or electron beams at a dose of 12.5-175 kGy in the presence of oxygen after manufacture. During the irradiation, the membrane may be in a dry or wet state and may be covered by air, water or an aqueous solution, respectively. The present invention also relates to a method of making such a membrane. The present invention also relates to a method for promoting cell adhesion and for using the membrane to culture cells, particularly adherent cells, without the need to pre-treat or precoat the membrane with any extracellular matrix components. Preferred membranes in the context of the present invention are polysulfone, polyether sulfone or poly (aryl) ether sulfone based synthetic membranes which also contain PVP and optionally a small amount of additional polymers such as polyamides or polyurethanes.

Brief Description of Drawings

Figure 1 shows the number [%] of MSCs grown from untreated bone marrow on various membrane types relative to the number of MSCs grown on standard TCPS after the first (14 days) and second (7 days) growth Experiment 1). The horizontal line represents the TCPS value. See Table I for the abbreviations used.

Figure 2 shows the number [%] of MSCs grown from untreated bone marrow on various membrane types relative to the number of MSCs grown on standard TCPS after the first (14 days) and second (7 days) growth Experiment 2). The horizontal line represents the TCPS value. See Table I for abbreviations used.

Figure 3 shows the form of the re-flushed MSC on a typical TCPS dish on the fifth day of the second growing season. The MSC is derived from Experiment 2 as described in Example 10. Abbreviations are given in Table I. (A) VTK membrane, water-covered, 0% acrylic acid, 75 kGy. (B) VTK membrane, water-covered, 0.000% acrylic acid, 75 kGy. (C) VTK membrane, water-covered, 0.001% acrylic acid, 75 kGy. (D) VTK membrane, water-covered, 0.01% acrylic acid, 75 kGy. (E) TCPS. (F) VTK membrane, air-covered, 25 kGy. (G) VTK membrane, air-covered, 75 kGy. (H) VTK membrane, untreated. (J) VTK membrane + FN coating.

Figure 4 shows the number [%] of MSCs grown from preselected MSCs on various membrane types relative to the number of MSCs grown on standard TCPS after the first (9 days) and second (7 days) growth (Experiment 1). The horizontal line represents the TCPS value. The abbreviations used refer to Table II and Example 11.

Figure 5 shows the number [%] of MSCs grown from pre-selected MSCs on various membrane types relative to the number of MSCs grown on standard TCPS after the first (10 days) and second (11 days) growth (Experiment 2). The horizontal line represents the TCPS value. The abbreviations used refer to Table II and Example 11.

Figure 6 shows a comparison of a direct gamma irradiated membrane and a steam sterilized membrane prior to gamma irradiation. Experiments as described in Example 12 relate to the number [%] of MSCs that adhere to the surface of a given membrane relative to the number of MSCs that adhere to a standard TCPS surface. The horizontal line shows the TCPS values.

Figure 7 shows the results of the untreated < RTI ID = 0.0 > untreated < / RTI > cells on irradiated 75 kGy with w / o steam and ETO sterilization, relative to the number of MSCs grown on standard TCPS after the first (11 days) And the number [%] of MSCs grown from bone marrow. The horizontal line represents the TCPS value.

Figure 8 illustrates the successful adipogenesis of MSCs grown on the membrane of the present invention. (A) VTK membrane, water-covered, 0.01% acrylic acid, 75 kGy. (B) VTK membrane, water-covered, 0.01% allylamine, 75 kGy. (C) VTK membrane, water-covered, 0.01% acrylic acid and 0.01% allylamine, 25 kGy.

Figure 9 illustrates successful incomplete osteogenesis of MSCs grown on the membrane of the present invention. (A) TCPS membrane (compare). (B) VTK membrane, water-covered, 0.01% acrylic acid, 75 kGy. (C) VTK membrane, water-covered, 0.01% allylamine, 75 kGy. (D) VTK membrane, water-covered, 0.01% acrylic acid + 0.01% allylamine, 75 kGy. (E) VTK membrane, water-covered, 0.01% acrylic acid, 25 kGy. (F) VTK membrane, water-covered, 0.5% acrylic acid, 25 kGy.

Figure 10 shows the results of short-term cell culture of human dermal fibroblast (NHDF) on VTK membranes irradiated by 25 kGy (air-covered) or 75 kGy (water-covered) compared to TCPS. The left column shows the number of NHDF cells after one day relative to the number of cells on the TCS, that is, the efficiency of cell adhesion. The right column shows the number of NHDF cells after 5 days relative to the number of cells on TCPS, i.e., it shows the efficiency of cell proliferation.

Figure 11 shows the results of short-term cell culture of human hepatocarcinoma cells (HepG2) on VTK membranes irradiated by 25 kGy (air-covered) or 75 kGy (water-covered) as compared to TCPS will be. The left column shows the number of HepG2 cells after one day relative to the number of cells on the TCPS, that is, the efficiency of cell adhesion. The right column shows the number of HepG2 cells after 5 days relative to the number of cells on the TCPS, that is, it shows the efficiency of cell proliferation. The membrane according to the present invention exhibits much better cell adhesion and proliferation than the TCPS surface.

Figure 12 shows the results of short-term cell culture of human kidney epithelial cells (HK-2) on VTK membranes irradiated by 25 kGy (air-covered) or 75 kGy (water-covered) . The left column shows the number of HK-2 cells after 1 day relative to the number of cells on the TCPS, that is, the efficiency of cell adhesion. The right column shows the number of HK-2 cells after 5 days relative to the number of cells on the TCPS, that is, it shows the efficiency of cell proliferation.

Figure 13 shows the results of short-term cell culture of canine epithelial cells (MDCK) on VTK membranes irradiated by 25 kGy (air-covered) or 75 kGy (water-covered) compared to TCPS. The left column shows the number of MDCK cells after one day relative to the number of cells on the TCPS, i.e., the efficiency of cell adhesion. The right column shows the number of MDCK cells after 5 days relative to the number of cells on TCPS, i.e., it shows the efficiency of cell proliferation.

Figure 14 shows the cell doubling time ratios in a membrane-based bioreactor according to the invention compared to standard flask cultures (see Example 15). The doubling time means the doubling time of the cells in a given bioreactor / doubling time of the cells in the control flask.

Figure 15 shows the viability of MSCs cultured from untreated bone marrow. The viability of the collected cells is over 90% for all bioreactors tested.

Figure 16 shows the phenotype of cells collected from a bioreactor according to the present invention. MSCs are cultured from untreated bone marrow. As can be seen, cells grown in VTK water-filled bioreactors (75 kGy) showed relatively high values for CD45 and HLA-DR. Cells collected from all other bioreactors represent the predicted MSC phenotype for CD34, CD45, CD73, CD90, CD105 and HLA-DR.

Figure 17 shows the number [%] of MSCs grown from pre-selected MSCs on various membrane types relative to the number of MSCs grown on standard TCPS after the first (10 days) and second (11 days) growth . The results show the effect of gamma dose on the cell proliferation characteristics of the membrane. The horizontal line represents the TCPS value.

DETAILED DESCRIPTION OF THE INVENTION

The object of the present invention is a polymer membrane which is treated in a dry or wet state with a dose of 12.5-175 kGy in the presence of oxygen by beta ray or gamma radiation or electron beam irradiation. The membrane may be surrounded by water, by water, or by an aqueous solution comprising a low amount of additive during irradiation, wherein oxygen in the air is present in the range of 4 to 100 vol%, such as 5 vol% To 30% by volume or from 15% by volume to 25% by volume.

The polymeric membrane comprises a hydrophobic or hydrophilic polymer, or both. In one embodiment, the membrane comprises a blend of one or more hydrophilic polymers and one or more hydrophobic polymers. In another embodiment, the membrane comprises a hydrophilic copolymer. In another embodiment, the membrane comprises a hydrophilic copolymer and a hydrophobic polymer. In another embodiment, the membrane comprises a hydrophilic homopolymer. In a further embodiment, the membrane comprises a hydrophilic homopolymer and a hydrophobic polymer.

In one embodiment of the present invention, the polymer used to prepare the membrane has a hydrophobic polymer and a hydrophilic polymer in an amount such that the fraction of hydrophobic polymer in the polymer solution is 5 to 20 wt% and the fraction of hydrophilic polymer is 2 to 13 wt% .

In a specific embodiment, the membrane comprises a first hydrophobic polymer component, a second hydrophilic polymer component, and optionally a third hydrophobic polymer component.

Wherein the first hydrophobic polymer is selected from the group consisting of polyamides (PA), polyaramides (PAA), poly (aryl) ether sulfone (PAES), polyethersulfone (PES), polysulfone (PSU), polyaryl sulfone (PC), polyether, polyurethane (PUR), polyetherimide, and copolymers of the foregoing polymers. Wherein the second hydrophilic polymer is selected from the group consisting of polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), polyglycol monoester, water soluble cellulose derivative, polysorbate and polyethylene oxide-polypropylene oxide copolymer desirable. The third hydrophobic polymer is selected from the group consisting of polyamides (PA), polyaramides (PAA), poly (aryl) ether sulfone (PAES), polyethersulfone (PES), polysulfone (PSU), polyaryl sulfone (PC), polyether, polyurethane (PUR), polyetherimide, and copolymers of the foregoing polymers. The membrane can be made in the form of a flat sheet or hollow fiber membrane.

In one aspect of the invention, the membrane may be useful for cell attachment or adhesion, cell growth and proliferation, or for storage of cells without the need for pre-treatment or precoating of the membrane by EMC. Advantages of using membranes without any such EMC in cell culture include lower cost, EMC (GMP-Compliance), or the greater number required to coat the membrane, for example in terms of time savings and fewer processing steps Lt; RTI ID = 0.0 > and < / RTI > significantly less well defined materials and protocols for cell production.

Of course, it is also possible to pre-treat or precoat the membrane of the present invention with one or more EMCs generally known in the art. In particular, for uses that are not intended to grow or grow cells or tissues for re-implantation into a host, such pretreatment can further improve the performance of the membrane in terms of adhesion or proliferation. However, it is always desirable to use the membrane of the present invention without any coating by EMC.

In a further aspect of the invention, performance in cell culture can be significantly improved by preparing the hollow fiber membranes of the present invention and by using the hollow fiber membranes or their bundles in a continuous culture process as an alternative to plate culture techniques . In addition to the continuous process, the hollow fiber membrane can be used in a static or semi-continuous process

The membranes of the present invention may be prepared in a manner that provides specific adhesion properties to the exterior and interior of hollow fiber membranes, as well as throughout the membrane, for example, for hollow fiber membranes for continuous applications.

In a further embodiment of the invention, the membrane also provides a system for cell co-cultivation of two or more different cell types.

A further aspect of the present invention is that the membrane promotes the formation of a most suitable cell monolayer in terms of differentiation or integrity without the need to precoat the membrane surface with any EMC. The membrane of the present invention provides retention of typical cell morphology, a single layer is easily formed, and a tight bond can be produced. In the context of the present invention, a monolayer refers to a layer of cells in which all of the cells are growing on both sides and, in many cases, are in contact with one another on the same growth surface, This is not necessarily the case for all potential uses.

Therefore, the membrane of the present invention can be used, for example,

(a) to tissue culture techniques, i.e. to achieve biosynthetic implants, such as biosynthetic kidneys or liver (also see Atala (2006));

(b) an MSC, a smooth muscle cell, a skin cell, a neuron, a systemic glia or an endothelial cell, for use in medical therapy via injection of a cell, which need to be proliferated in vitro, , Or floating cells, such as hematopoietic stem cells, cord blood cells, neural stem cells, and the like,

(c) for propagating and providing cells that act as producers of growth factors, recombinant proteins, cytokines or bio-products such as antibodies, such as monoclonal antibodies,

(d) for culturing adherent cell cultures, preferably for cell monolayer cultures, for studying specific cell types, or for treating any cell mediated drugs, such as anticancer, antimicrobial, anti- (Screening procedures) for studying the effects of biotics, antiviral agents (including anti-HIV viruses) or anti-parasitic agents, or

(e) on any other use that is based on the culture or storage of the adhered or floating cells in an in vitro system or which requires its growth or culture

Can be advantageously used.

The membrane of the present invention may have any suitable geometry depending on the needs of the intended application, i.e. it may be a flat sheet, hollow fiber, or hollow fiber bundle, or may be shaped to form a chamber or other desired geometry . The core unit for cell proliferation is preferably a hollow fiber based membrane system that allows for sufficient exchange of O ₂ and CO ₂ , supply of nutrients and removal of waste products. The surface of the membrane is designed to enable adhesion and proliferation of cells with certain characteristics through specific surface features. Advantages of cell culture inside hollow fibers are based on advantageous surface-to-volume ratio that results in minimization of media consumption, minimization of space requirements and minimization of labor in culture processes as compared to conventional flasks or cell stack culture methods One point. Another advantage of the hollow fiber structure is the uniformly controlled flow path.

The membranes of the present invention can be used in various types of cell proliferation or cell culture devices or systems as described, for example, in US 2003/0203478 Al, US 6,150,164 or US 6,942,879.

The membrane of the present invention can generally be used advantageously to cultivate adherent cells. Adherent cells are defined in the context of the present invention as cells adhering to the substrate to be maintained, proliferated, differentiated, stored, and the like. The membrane of the present invention is useful for culturing embryonic stem cells and adult stem cells, particularly stem cells such as mesenchymal stem cells (MSC), fibroblasts, epithelial cells, hepatocytes, endothelial cells, muscle cells and cartilage cells Can be used.

In the first aspect of the present invention, the membrane of the present invention has a hydrophobic polymer in an amount such that the fraction of the hydrophobic polymer in the polymer solution used to prepare the membrane is 5 to 20 wt% and the fraction of the hydrophilic polymer is 2 to 12 wt% And a hydrophilic polymer. Wherein the at least one hydrophobic polymer is selected from the group consisting of polyamides (PA), polyaramides (PAA), polyarylether sulfone (PAES), polyethersulfone (PES), polysulfone (PSU), polyaryl sulfone (PC), polyether, polyurethane (PUR), polyetherimide and copolymers of such polymers, preferably polyethersulfone or mixtures of polyarylether sulfone and polyamide. Wherein the at least one hydrophilic polymer is selected from the group consisting of polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), polyglycol monoester, water soluble cellulose derivative, polysorbate and polyethylene-polypropylene oxide copolymer, preferably polyvinylpyrrolidone ≪ / RTI >

In the context of the present invention, a membrane that can preferably be used is a membrane which is selected from the group consisting of polysulfone (PS), polyethersulfone (PES) and polyarylether sulfone (PASES) in a polymer solution for making the membrane From 0 to 5% by weight, preferably from 0.001 to 5% by weight of polyamide (PA), from 0 to 30% by weight of water, and from 0 to 5% by weight of a second polymer such as polyvinylpyrrolidone (PVP) (NNP), preferably N-ethyl-2-pyrrolidone (NEP), N-octyl-2-pyrrolidone (NOP), dimethylacetamide Up to 100 wt% of a solvent selected from the group consisting of dimethylformamide (DMF), dimethylsulfoxide (DMSO), and gamma-butyrolactone (GBL).

Preferably, the polyvinylpyrrolidone (PVP) in the polymer solution is composed of a blend of at least two homopolymers of polyvinylpyrrolidone, wherein one of the homopolymers of polyvinylpyrrolidone (= low molecular weight PVP ) Has an average relative molecular weight of from about 10,000 g / mol to 100,000 g / mol, preferably from about 30,000 g / mol to 70,000 g / mol, and the remaining one of the homopolymers of polyvinylpyrrolidone (= high molecular weight PVP) Has an average relative molecular weight of about 500,000 g / mol to 2,000,000 g / mol, preferably about 800,000 g / mol to 2,000,000 g / mol. Examples of such PVP homopolymers are PVP K85, high molecular weight PVP having a molecular weight of about 825,000 Da and PVP K30, and low molecular weight PVP having a molecular weight of about 66,800 Da. In a preferred embodiment of the present invention, the polymer solution for making the membrane comprises 0.5 to 5% by weight of high molecular weight PVP and 1 to 8% by weight of low molecular weight PVP.

Methods for making such membranes are described in detail in, for example, US-A 4,935,141, US-A 5,891,338 and EP 1 578 521 Al, all of which are incorporated herein by reference. Examples of this type of membrane that can be effectively treated in accordance with the present invention include Gambro Polyflux ™ membranes (polyarylether sulfone / PVP / polyamide), which are currently used in commercial products such as Polyflux ™ L and H series has exist); Arylane ™ Membrane (poly (aryl) ether sulfone / PVP); Or other commercial dialysis membranes based on blends of DIAPES (TM) or PUREMA (TM) membranes (poly (aryl) ether sulfone / PVP) or blends of hydrophilic polymers with hydrophobic polymers such as PVP and PES or polysulfone.

In a second aspect of the invention, the polymer solution used to prepare the membrane of the present invention comprises 12 to 15% by weight of polyethersulfone or polysulfone as hydrophobic polymer and 5 to 10% by weight of PVP, Molecular weight PVP component and high molecular weight PVP component. The total PVP contained in the spinning solution has a high molecular weight (> 100 kDa) component of 22 to 34 wt%, preferably 25 to 30 wt%, and a low molecular weight (≤ 100 kDa) components. Examples of high molecular weight and low molecular weight PVP are, for example, PVP K85 / K90 and PVP K30, respectively. Preferably, the polymer solution used in the process of the present invention further comprises from 66 to 86% by weight of solvent and from 1 to 5% by weight of suitable additives. Suitable additives include, for example, water, glycerol and / or other alcohols. Water is particularly preferred and, when used, is present in the spinning solution in an amount of from 1 to 8% by weight, preferably from 2 to 5% by weight. The solvent used in the process of the present invention is selected from a mixture of N-methylpyrrolidone (NMP), dimethylacetamide (DMAC), dimethylsulfoxide (DMSO), dimethylformamide (DMF), butyrolactone, . NMP is particularly preferred. The center fluid or bore liquid used to make the membrane preparation comprises at least one of the above-mentioned solvents and a precipitation medium selected from water, glycerol and other alcohols. Most preferably, the central fluid is comprised of 45 to 70 wt% of precipitation medium and 30 to 55 wt% of solvent. Preferably, the center fluid consists of 51 to 57 wt% water and 43 to 49 wt% NMP.

Methods for making such membranes are described in detail in European Patent Application No. 08008229, which is expressly incorporated herein by reference. Examples of such membranes that can be effectively treated in accordance with the present invention are, for example, Gambro Revaclear ™ membranes and their derivatives. Also, in the context of the present invention, blends of hydrophobic polymers with membranes or hydrophilic polymers currently used in commercial products such as, for example, Fresenius FX ™ -brained membranes (Helixone ™ membranes) or Optiflux ™ -type membranes, It is also possible to use other commercial dialysis membranes based on a blend comprising PES or polysulfone.

In a third aspect of the invention, the polymer solution used to prepare the membrane of the present invention comprises a first polymer 11 selected from the group consisting of polysulfone (PS), polyethersulfone (PES) and polyarylethersulfone (PAES) (PU), 0 to 7% by weight of water, and 1 to 5% by weight of a second polymer such as polyvinyl pyrrolidone (PVP) (NMP), N-ethyl-2-pyrrolidone (NEP), N-octyl-2-pyrrolidone (NOP), dimethylacetamide, dimethylformamide (DMF), dimethylsulfoxide -Butyrolactone (GBL). ≪ / RTI > The first polymer is preferably present in the polymer solution in an amount of 13 to 14% by weight, particularly preferably in an amount of 13.6 to 14% by weight. It is preferred that polyethersulfone (PES) and polyarylethersulfone (PAES) are used to prepare the membrane of the present invention. The polyvinylpyrrolidone (PVP) in the polymer solution is preferably composed of a blend of at least two homopolymers of polyvinylpyrrolidone, wherein one of the homopolymers of polyvinylpyrrolidone (= (low molecular weight PVP ) Has an average relative molecular weight of from about 10,000 g / mol to 100,000 g / mol, preferably from about 30,000 g / mol to 70,000 g / mol, and another one of the homopolymers of polyvinylpyrrolidone (= high molecular weight PVP ) Has an average relative molecular weight of from about 500,000 g / mol to about 2,000,000 g / mol, preferably from about 800,000 g / mol to about 2,000,000 g / mol. Examples of such homopolymers include PVP K85 having a molecular weight of about 825,000 Da And PVP K30, a low molecular weight PVP having a molecular weight of about 66,800 Da. In a preferred embodiment of the present invention, the polymer solution for making the membrane comprises 0.5 to 5% by weight of high molecular weight PVP and 1 to 5% 8 wt% The water content of the spinning solution is preferably 1 to 5% by weight, more preferably about 3% by weight. Various solvents such as N-methyl-2-pyrrolidone (NMP), N- N-octyl-2-pyrrolidone (NOP), dimethylacetamide, dimethylformamide (DMF), dimethylsulfoxide (DMSO) or gamma-butyrolactone (GBL) And mixtures thereof can be used to prepare the membranes of the present invention The solvent is present in an amount of up to 100% by weight of the polymer solution The content of solvent in the polymer solution is preferably 60 to 80% by weight, Is 67 to 76.4% by weight.

The membrane of the present invention can be made, for example, as a flat sheet or hollow fiber geometry.

In one embodiment, the membrane of the present invention has an asymmetric structure. In the case of hollow fibers, there is a thin separation layer on the inner surface of the fibers. The structure or morphology of the membrane of the present invention may be varied without significant effect on the performance of the cell adhesion and proliferation. The membrane may have, for example, a three-layer structure or a sponge-like structure or a foam-like structure. In one embodiment, the membrane of the present invention is further characterized by the smoothness or low roughness of the cell adhesion surface.

In one embodiment, the hydraulic permeability of the membrane of the present invention (hydraulic permeability) is about 0.1 and 10 ^-4 cm ³ to 200 and ^{10 -4 cm 3 / (cm 2} bar sec), for example, 0.1 and 10 ^-4 cm ³ to 10 10 ^-4 cm ³ / (cm ² bar sec), or even 0.1 10 ^-4 cm ³ to 5 10 ^-4 cm ³ / (cm ² bar sec). The viscosity of the polymer solution is usually in the range of 2,500 cP to 200,000 cP, or even 10,900 cP to 25,600 cP for the preparation of the filamentary fibers, in order to achieve such water pressure transmittance without obtaining defects in the membrane structure. For flat sheet membrane fabrication, the viscosity is generally in the range of from 2,500 cP to 500,000 cP, or even 4,500 cP to 415,000 cP.

When preparing the membrane of the present invention, the polymer is dissolved in the solvent at a constant temperature and pressure. The degassing process of the polymer solution is carried out in a drying oven producing a vacuum (approximately 100 mbar). The temperature of the polymer solution may vary over a relatively wide range. It is advantageous to select a temperature within the range of room temperature to 60 deg.

When producing a flat sheet membrane, the final polymer solution is cast as a uniform film on a smooth surface, such as a glass slide, that serves as a support area, by using a special coating knife. The rate at which the polymer film is cast may vary over a relatively wide range. A speed of 10 to 20 mm / s may be suitable. In an exemplary lab scale process, the polymer solution is first applied steadily on a glass slide using a syringe. It is important to work without bubbles. Coated knives with limited gap heights act at a constant rate, which results in a uniform polymer film. For an excellent thickness distribution, a coated knife having a uniform gap is preferred.

In one embodiment of the present invention, the settling vessel comprises H ₂ O in an amount of 30 to 100 wt%, preferably 56 to 66 wt%, and 0 to 70 wt%, preferably 34 to 44 wt% %, ≪ / RTI > such as NMP. The temperature of the settling vessel can vary over a relatively wide range. It may be advantageous to apply a temperature of 0 ° C to 80 ° C, or 30 ° C to 50 ° C. The settling time can also vary. As an example, the settling time can be about 5 minutes. The precipitation bath is preferably composed of H ₂ O and a solvent. The precipitating bath comprises N-methyl-2-pyrrolidone (NMP), N-ethyl-pyrrolidone (NMP), and mixtures thereof, present in an amount of 30 wt.% To 100 wt.% H ₂ O and 70 wt. (NEP), N-octyl-2-pyrrolidone (NOP), dimethylacetamide, dimethylformamide (DMF), dimethylsulfoxide (DMSO), or gamma-butyrolactone And mixtures thereof. In one embodiment of the present invention, the precipitation vessel comprises H ₂ O present in an amount of 56 to 66 wt%, and a solvent present in an amount of 34 to 44 wt%. NMP is a particularly suitable solvent in the context of the present invention.

The precipitated membrane is then stored in the nonpolymer until the membrane is broken. After cleavage, the membrane is washed, dried and sterilized.

The thickness of the flat sheet membrane of the present invention may vary from 15 占 퐉 to 200 占 퐉. Thicknesses from 35 μm to 50 μm may be particularly advantageous for most applications.

The membranes of the present invention may also be fabricated into hollow fiber geometries. When making such hollow fiber membranes, the solution is pumped through a spinning die, and liquid hollow fibers are formed. The solvent concentration at the center results in an open structure at the inner surface of the membrane. The smallest pores are formed directly on the inner surface of the membrane. In use, the optional layer is in direct contact with the cell culture medium.

In one embodiment, the settling tank is comprised of water. The temperature of the settling vessel may vary over a wide range, but an ambient temperature to about 40 캜 is advantageously used in the process. The distance between the die and the settling tank is in the range of 0 to 100 cm, for example, 50 to 100 cm. The die (spinneret) temperature can also vary. Temperatures of 20 to 80 占 폚 may be used. It may be advantageous to apply a temperature of 40 to 50 캜. The spinning speed can be selected within the range of 5 to 80 m / min, for example 11 to 40 m / min.

The dimensions of the hollow fiber membrane of the present invention may vary depending upon the intended use of the membrane. The internal diameter is generally in the range of 50 to 2,000 μm. For many applications, an internal diameter of 100 to 950 占 퐉 may be advantageous. The overall thickness is generally in the range of 25 to 55 μm.

Lp provided as a result of the membrane of the present invention is 0.1 to 200 and 10 ^-4 and 10 ^-4 cm ³ / (cm ² bar and and s), for example, 0.1 and ^1-4 to 10 and 10 ^-4 cm ³ / (cm < ² > bar s), or even in the range of 0.1 10 ^-4 to 5 10 ^-4 cm ³ / (cm ² bar s). In another embodiment of the present invention, the Lp of the membrane is in the range of 2 · 10 ^-4 to 18 · 10 ^-4 cm ³ / (cm ² · bar · s). In another embodiment of the present invention, the Lp of the membrane is in the range of 5 · 10 ^-4 to 15 · 10 ^-4 cm ³ / (cm ² · bar · s). That is, the membrane to be used for cell culture may be a so-called low flux membrane.

There are two ways to produce the membranes of the present invention, which can be referred to as "wet" and "dry ". When a "wet" membrane is produced, the membrane must be dried separately in tubes or ovens after manufacture. For this purpose, a bundle of fibers (for example, 30 to 15,000 fibers) is disposed in a plastic or metal container. Hot air is passed through the vessel to dry the membrane. The second approach is an "on-line drying" which is an efficient way to directly produce dried hollow fibers on a spinning machine. Both procedures are applicable to achieve a membrane that can be processed and used in accordance with the present invention.

According to the present invention, a membrane as described above is characterized in that the membrane is treated with air or water or an additive such as acrylic acid, allylamide or acrylamide in an amount of from 0.00001% to 5% by weight, for example from 0.0001% to 0.01% And treated with gamma, beta or electron beam irradiation in the presence of oxygen.

In order to achieve a membrane that can serve for cell culture purposes, the membrane is placed in an irradiation chamber and treated with gamma, beta or electron beam irradiation, in particular with gamma irradiation, using an irradiation dose of 12.5-175 kGy, preferably 70-175 kGy can do. In another embodiment of the present invention, the dose used is 25 to 125 kGy. In another embodiment of the invention, a dose of 50-175 kGy is used. In another embodiment of the invention, a dose of 50 to 125 kGy is used. In another embodiment of the invention, a dose of 70-100 kGy is used.

Gamma irradiation can be performed, for example, using a Co-60 source. The electron beam irradiation can be performed using an electron beam accelerator. Beta radiation can be performed using a beta radiation source, such as Sr-90 or Ru-106.

In one embodiment of the present invention, the membrane is irradiated under dry conditions, i.e. the membrane is covered with air or surrounded by air. In the context of the present invention, the expression "dry" does not exclude the presence of water in the porous structure of the membrane, that is to say within the range from the complete member of water to the condition that the completely porous structure of the membrane wall is filled with water, do.

For example, in order to sterilize the membrane or to cross-link certain components of the membrane, for example PVP, the presence of oxygen during irradiation, in contrast to the known gamma-rays of the membrane, . The ambient air during the irradiation is, in one embodiment of the invention, approximately 78.08% nitrogen, 20.95% oxygen, 0.93% argon, 0.038% carbon dioxide, trace amounts of other gases, and approximately varying amounts of water vapor (molar content / Or the like. In another aspect of the present invention, the oxygen content of the ambient air can be increased, for example, by additionally introducing oxygen gas into the system. The oxygen concentration can be increased to a limit value of about 100%, for example up to 30%. In another embodiment of the present invention, the oxygen concentration can be lowered to a limit of about 4%. 4% to 100%, for example 4% to 30% (molar content / unit of volume) can generally be used to achieve the desired result. It may be advantageous to use a concentration of oxygen between 5% and 25%, or even between 15% and 22%.

In another embodiment of the present invention, the membrane is irradiated under wet conditions, or in other words, under aqueous conditions. The expressions "wet" and "aqueous conditions" mean, in the context of the present invention, the presence of water during the course of the irradiation, i.e. the membrane can be covered with water or impregnated in water. In one embodiment of the present invention, RO water is used.

In another embodiment of the present invention, the additive may be mixed with water in a low amount. Such additives can improve the performance of membranes typically irradiated for cell culture purposes or on certain cell types by introducing functional groups on the membrane surface. Examples of such additives include vinyl group containing monomers having an amino, carboxyl or carboxamide functional group such as acrylic acid, allylamide or acrylamide. In one specific embodiment, allylamide is used. The additive may be present in the aqueous solution at a concentration of 0.00001% to 5% by weight. It may be advantageous to use a concentration of 0.0001% to 1% by weight, or 0.0001% to 0.1% by weight. Low concentrations of additives, for example from 0.0001% to 0.01% by weight, can be particularly effectively demonstrated.

It may be advantageous to use a higher dose of radiation, e. G. 70 to 175 kGy, for irradiation of the membrane in the wet state.

The time required to achieve the selected dose can vary over a relatively wide range. As an example, using a Co-60 source may require about 6 to 7 hours for a 25 kGy dose and about 17 to 20 hours for a 75 kGy dose, i.e., the irradiation time is 3 times. The time required depends on the source itself and on the intensity and the need to adjust it.

The temperature can also vary over a wide range and also depends on the material of the housing for the membrane, i.e. when the housing is made of a metal or a synthetic material, the temperature is generally within the range of 0 ° C to 41 ° C have. In most cases, it is easy to use room temperature.

In another embodiment of the present invention, the membrane is dried, preferably on-line drying, by easter steam sterilization treatment, prior to irradiation, in order to retain the desired low flux characteristics. Additional sterilization treatment with EtO (ethylene oxide) may be added without adversely affecting the efficiency of the membrane of the present invention with respect to its use for cell culture purposes if so desired or necessary in the manufacturing process. Methods of steam sterilization or EtO sterilization of membranes are well known in the art.

The irradiated membrane can then be used directly to culture different types of cells, preferably cohesive cells. The membranes of the present invention exhibit substantially similar or even better growth characteristics to tissue culture polystyrene (TCPS) plates, which present a current gold standard for cell proliferation using culture flasks or cell stacks.

The membranes of the present invention can be used in tissue culture polystyrene (TCPS) as shown in the cell growth rate, cell reattachment efficiency on the membrane, and tests performed by mesenchymal stem cells (MSC), fibroblasts, epithelial cells and hepatocytes. Lt; RTI ID = 0.0 > and / or < / RTI > very good morphology.

A further aspect of the invention is a cell culture apparatus comprising the membrane of the invention. Examples of cell proliferation or cell culture apparatus or systems that can be modified to include the membrane of the present invention are disclosed in US 2003/0203478 Al, US-A 6,150,164, or US-A 6,942,879, all of which are incorporated herein by reference . The apparatus may comprise a stack of flat sheet membranes of the present invention or a bundle of hollow fiber membranes of the present invention.

In one embodiment of the apparatus, the membrane forms an interface between two fluid compartments of the apparatus. The device may be similar in construction to, for example, a commercially available filtration device used for hemodialysis or blood filtration.

An exemplary apparatus includes two compartments separated by a semipermeable membrane enclosed within a casing, wherein the first interior compartment is adapted by two actuators, the second exterior compartment comprises one or two actuators, The proton compartments may also be of any type, including, but not limited to: (i) a threaded partition that separates the compartments of both of the devices containing a semipermeable membrane of the hollow fiber bundle type as defined above; or (ii) a sheet membrane type As a gas tight seal in the device comprising a semipermeable membrane of the invention, also separated by a potting compound based on a suitable adhesive compound intended to form.

Other exemplary devices include a plurality of hollow fiber membranes contained within the outer shell and configured such that the fluid in the hollow fiber outer space (i.e., the outer capillary section) is isolated from the fluid through which the hollow fibers and corresponding orifices pass . Additionally, the apparatus includes two manifolds and chambers in an outer shell on the opposite end of the apparatus. Each of the two openings of the hollow fiber is connected to a different end chamber. The end chamber and capillary outer compartment are separated by semi-permeable membranes of hollow fibers. The composition within the capillary outer compartment can be controlled to a certain degree, by the molecular weight cutoff or pore size of the membrane of the hollow fibers.

In one way to operate the device, cells are grown in the capillary outer compartment, passing the nutrient medium through the hollow fibers. The medium can pass through the capillary or through the capillary compartment. In another manner of operating the device, the cells are grown in the capillary space (i.e., lumen) of the hollow fibers while passing nutrient media through the capillaries and / or through the capillary compartments. The semipermeable nature of the hollow fiber is to allow nutrients, gases and cell waste products to pass through the walls of the hollow fibers, while at the same time blocking the cells from doing so.

Shell-and-tube type bioreactors offer several advantages. In the case of adherent cells, the use of several hollow fibers provides a large surface area within which cells can grow, within relatively small volumes. This large amount of surface area also facilitates localized distribution of nutrient media to the growing cells and immediate collection of cell lung products. Shell and tube type bioreactors enable cell growth at a much higher density rate than is possible by other cell culture devices. The bioreactor can support a cell density of more than 10 ⁸ cells per milliliter, while other cell culture devices are typically limited to a density of about 10 ⁶ cells per milliliter.

A further aspect of the invention provides an apparatus for in vitro treatment of cells and body fluids comprising the inventive membrane. In one embodiment, the cell has a confluent layer on the surface of the membrane, for example on the surface of the lumen of the hollow fiber membrane of the present invention or on the outer surface of the hollow fiber of the present invention, Of the cells. For the remainder, the design of the device may be similar to the design described above for the cell culture device. The bodily fluid to be treated is conducted through the fluid space of the device, wherein the fluid passes over the cell layer, allowing the cell to extract the component from the bodily fluid, metabolize the component of the bodily fluid, or isolate the component into the bodily fluid.

Example

The evaluation of the suitability and efficiency of the membranes of the present invention was generally based on the following key features: cell proliferation rate, cell re-adhesion efficiency on the membrane, and post-proliferation characteristics including morphological control. The tests were performed with mesenchymal stem cells (MSC), fibroblasts, epithelial cells and hepatocytes to demonstrate that the membranes according to the invention are suitable for culturing a variety of coalesced cell types. MSCs were selected for depth analysis of the membrane performance of the present invention for cell culture.

Way

hand  Bundles, mini modules, filters and flat bedsheet  Manufacture of inserts

(A) hand  bunch

The manufacture of membrane bundles after the spinning process is required to prepare the fiber bundles in a manner suitable for successful performance testing. In the first process step, the fiber bundle is cut to a defined length of 23 cm. The next process step consists of melting the ends of the fibers. Optical control ensures that all fibers are well melted. The end of the fiber bundle is then transferred into the potting cap. The potting cap is mechanically fixed and the potting tube is placed over the potting cap. Thereafter, the potting is performed with polyurethane. The potting should be performed so that the polyurethane cures for at least one day. The final process step consists of optical control of fiber bundles. During this process step, the following are selected: (i) the quality of the cut (whether the cut is smooth or any damage to the knife), (ii) the quality of the potting (the open fiber of the spinning process reduced by the potting fiber Whether there is any visible void or void free of polyurethane). After optical control, the membrane bundles are stored dry prior to use in different performance tests.

(B) Manufacturing of mini-modules

A mini module [= fiber bundle in the housing] is manufactured by the associated process steps. This mini module is necessary to protect the fiber and ensure a very clean manufacturing process. The manufacturing process of the mini-module differs from: (i) cutting the fiber bundle to a limited length of 20 cm, (ii) transferring the fiber bundle into the housing before the melting process, and (iii) In a vacuum drying oven.

(C) Production of filter

The filter comprises about 8,000 to 15,000 fibers with an effective surface area of 0.9 to 1.7 m < ^{2 &} gt ;. The filter is characterized by a cylindrical housing having two connectors for feeding the culture medium fluid and a cap applied to both, with one connector at the center each. (After wading) The manufacturing process involves the following major steps: (i) transferring a cut (about 30 cm in length) bundle into a housing by means of a special bundle claw, (ii) (Iii) potting the fibers into the housing by means of polyurethane (PUR), (iv) cutting the ends to open the fibers (here requiring a smooth surface), (v) Visually inspecting the fibers or incompleteness in the PUR block, (vii) performing final treatment: cleaning, integrity testing, final drying, (viii) additional steps (e.g., irradiation) And a step of packaging in a special bag.

(D) flat bedsheet  Manufacture of inserts

A flat membrane is fixed on the glass plate. The polyurethane acting as glue for the insert is evenly distributed on the plate. The insert is lightly soaked in polyurethane and immediately attached to each membrane with glue. The inserts are weighed with glass and iron plates and dried for 16-18 hours. The flat membrane insert is cut and placed into a sterilization bag for welding. Finally, the insert can be sterilized in an autoclave at < RTI ID = 0.0 > 121 C. < / RTI >

hand  Hydraulic transmittance of bundle and mini module ( Lp : Hydraulic Permeability )

The water pressure transmittance of the membrane bundle is determined by pressing the exact defined volume of water under pressure through a sealed bundle of membranes and measuring the time required. The water pressure transmittance can be calculated from the determined time, membrane effective surface area, applied pressure and volume of pressurized water passing through the membrane. The effective surface area of the membrane is calculated from the number of fibers, fiber length as well as the inner diameter of the fibers. Membrane bundles should be wet for 30 minutes before performing the Lp test. For this purpose, the membrane bundle is placed in a box containing 500 ml of ultrapure water. After 30 minutes, transfer the membrane bundle to the test system. The test system consists of a water tank, which is controlled at 37 ° C, and a device capable of mechanically carrying the membrane bundle. The filling height of the tank should ensure that the membrane bundle is located below the surface of the water in the designated device. In order to avoid leakage of the membrane resulting in erroneous test results, the integration test and test system of the membrane bundle must be performed in advance. Integrity testing is performed by pressurizing air so that one side of the membrane passes through the membrane bundle that is closed. Air bubbles indicate leakage of the membrane bundle and the test apparatus. It should be checked whether the leakage can be related to the misoperation of the membrane bundle in the test apparatus or if actual membrane leakage appears. The membrane bundle should be discarded if leakage of the membrane bundle is detected. The pressurized pressure of the integrity test should be at least equal to the pressure applied during the measurement of the water pressure permeability, in order to ensure that any leakage can not occur due to the pressure applied too high during the measurement of the water pressure permeability.

hand  Diffuse transmittance of bundle

Diffusion experiments with an isotonic chloride solution as well as diluted phosphate (100 mg / l) in a dialysis fluid are performed to determine the diffusion characteristics of the membrane. Place the hand bundle in the measuring cell. This measuring cell allows passage of a specific solution inside the hollow fiber. Additionally, the measuring cell is fully filled with water, and the high cross-flow of the distilled water is set to carry specific ions passing through the membrane cross-sectional area from the inside to the outside of the hollow fiber. By precisely adjusting the pressure ratio, zero filtration is achieved, measuring only the diffusion characteristics of the membrane (by achieving the maximum concentration gradient of the specific ion between the interior of the hollow fibers and the periphery of the hollow fibers), and the combination of diffusion and convection properties It is not measured. A sample derived from the pool is taken at the start and a sample of the retention is taken at 10 and 20 minutes. The chloride sample is then titrated with a silver nitrate solution to measure the chloride ion. Analyze the phosphate sample with a photometer. From the measured concentration, the membrane effective surface area A and the flow conditions, the permeability P of each chloride or phosphate can be calculated according to the following equation (2).

Px [10 ^-4 cm / s] = [Q _B / 60 / A] * ln [(C _A -C _D ) / C _R ] * 10 ⁴ (2)

In the formula,

P = diffusion transmittance [cm / s]

c = concentration [mmol]

A = membrane effective surface area [cm ² ]

 Indices:

x = substance (here, chloride or phosphate, respectively)

A = starting concentration (feed)

D = dialysate

R = retention

QB = blood flow [ml / min]

( hand  Lt; RTI ID = 0.0 > () < / RTI > sieving coefficient )

The extinction coefficient experiments in aqueous solutions of myoglobulin and albumin are carried out using two different experimental setups with separate solutions. As a first test, the constitution coefficient of myoglobin is measured.

The concentration of dissolved myoglobin in the PBS buffer is 100 mg / L. The end date of the aqueous solution is 4 to 8 weeks. The solution should be stored in the refrigerator. Before the extinction coefficient experiment, the Lp-test is performed using the method described earlier. (Jv, cm / s) and wall shear rate (γD, s- ¹ ) are fixed, while the blood flow rate (Q _B ) and the filtration rate UF (see equation (4) + (5)) and A is calculated according to equation (1).

QB [ml / min] = γ * n * π * di 3 * 60/32 (4)

UF [ml / min] = Jv * A * 60 (5)

here,

n = amount of fiber

d _I = inner diameter of fiber [cm]

γ = shear rate [s ^-1 ]

A = membrane effective surface area [cm ² ]

In order to test the hand bundle and the mini module, the shear rate is set to 500 s ^-1 and the intrinsic flow rate is defined as 0.38 揃 10 ^-04 cm / s.

The first sample is taken after 15 minutes (pool, retentate and filtrate) and taken a second after 60 minutes. At the end, the test bundle is rinsed with PBS buffer for a few minutes and then the test is stopped.

Example  One

flat bedsheet Mebrain  Produce

The polymer solution was prepared by dissolving distilled water in distilled water as well as polyethersulphone (Ultrason TM 6020, BASF), polyvinylpyrrolidone (K30 and K85, BASF), polyurethane (Desmopan TM 9665 DU, Bayer MaterialScience AG) Methylpyrrolidone (NMP) at room temperature to dissolve the solution until a transparent high-viscosity solution was obtained.

The weight fraction of the different components in the polymer spinning solution, PES / PVP (K85) / PVP (K30) / 9665DU / H 2 O / NMP was 14/3/5/2/3/73. The viscosity of the resulting polymer solution was 21,000 mPa s.

The solution was filtered and degassed. The degassing process of the polymer solution was carried out in a drying oven at elevated temperature (<100 ° C) and reduced pressure (approximately 100 mbar). The final polymer solution was then (automatically) cast as a uniform film on a smooth surface (glass slide) that served as a support area using a special coating knife. First, the polymer solution was heated in an oven to 60 占 폚 and then applied uniformly onto a glass slide using a syringe. A coating knife with a limited height of the gap (lOO [mu] m) was operated at a constant speed of 12.5 mm / s to form a uniform polymer film. Such a glass slide with a polymer film was rapidly immersed into the coagulation bath. As a coagulation tank, a water / NMP mixture containing 56% by weight of water and 44% by weight of NMP was used at 50 占 폚. The precipitation of the membrane took about 5 minutes. The precipitated membrane was then removed, and all membranes in the series were stored in the non-solvent until they were made, and then cut to a defined size. After cleavage, the membrane was washed with distilled water for 30 minutes at 70 &lt; 0 &gt; C. The following steps were performed in a drying step performed in an oven at 60 ° C overnight, and finally in a special bag used for sterilization treatment. The membrane thickness was 35 μm.

Example  2

flat bedsheet Membrane  Produce

The polymer solution was prepared by adding distilled water as well as polyethersulfone (Ultrason (R) 6020, BASF), polypyrylpyrrolidone (K30 and K90, BASF) Pyrrolidone (NMP).

The weight fraction of the different components in the polymer spinning solution, PES / PVP (K90) / PVP (K30) / H 2 O / NMP was 13.6 / 2/5/3 / 76.4. The resulting polymer had a viscosity of 5,900 mPa s.

The membrane was prepared as described in Example 1. The membrane thickness was 50 μm.

Example  3

Hollow fiber Membrane  Produce

The polymer solution was prepared by mixing 13.5 wt% polyethersulfone, 0.5 wt% polyamide, 7.5 wt% PVP K30, and 78.5 wt% NMP. A mixture of 59 wt% water and 41 wt% NMP served as the center fluid. The viscosity of the polymer solution was 4,230 mPa 측정 when measured at a temperature of 22 캜.

The center fluid was heated to 55 占 폚 and pumped through the bicomponent hollow fiber spinneret. The polymer solution was discharged from spinnerets through an annular slit having an outer diameter of 0.5 mm and an inner diameter of 0.35 nm. The center fluid was discharged from the spinneret at the center of the annular polymer solution tube to initiate precipitation of the polymer solution from the inside and measure the inner diameter of the hollow fiber.

At the same time, the two components (polymer solution and center fluid) were introduced into the space separated from the laboratory atmosphere. This space is called a spinning shaft. A mixture of steam (100 DEG C) and air (22 DEG C) was injected into the spinning shaft. The temperature in the spinning shaft was adjusted to 49 캜, where the relative humidity of 99.5% was adjusted to 3.9% by weight with respect to the water content by the ratio of steam and air and solvent content. The solvent was NMP. The length of the spinning shaft was 890 mm. With the aid of gravity or motor-driven rollers, hollow fibers were drawn from the spinneret through the spinning shaft in a vertical direction into the water tank from the top to the bottom. The spinning speed was 50 m / min. The hollow fibers were then induced through a multistage water bath and a temperature increasing from 20 占 폚 to 90 占 폚. The wet hollow fibers discharged from the washing bath were dried in a convection type on-line drying step. After the optional texturing step, the hollow fibers were collected on a bundle shaped spinning wheel. The outer surface of the hollow fibers according to this example had 62,500 pores per mm ² with pore diameters ranging from 0.5 to 3 μm.

Example  4

Hollow fiber Membrane  Produce

The polymer solution was prepared by mixing 14.0% by weight of polyethersulfone, 5.0% by weight of PVP K30, 2.0% by weight of PVP K85 / K90, 3% by weight of water and 76.0% by weight of NMP. A mixture of 55 wt% water and 45 wt% NMP served as the center fluid. The polymer solution had a viscosity of 5,400 mPa 측정 as measured at a temperature of 22 캜.

The center fluid was heated to 55 占 폚 and pumped through the bicomponent hollow fiber spinneret. The polymer solution was discharged from spinnerets through an annular slit having an outer diameter of 0.5 mm and an inner diameter of 0.35 nm. The center fluid was discharged from the spinneret at the center of the annular polymer solution tube to initiate precipitation of the polymer solution from the inside and measure the inner diameter of the hollow fiber. At the same time, the two components (polymer solution and center fluid) were introduced into the space separated from the laboratory atmosphere. This space is called a spinning shaft. A mixture of steam (100 DEG C) and air (22 DEG C) was injected into the spinning shaft. The temperature in the spinning shaft was adjusted to 45 캜, and a relative humidity of 99.5% was obtained therefrom by the ratio of steam and air and solvent content. The length of the spinning shaft was 890 mm. With the aid of gravity or motor-driven rollers, hollow fibers were drawn from the spinneret through the spinning shaft in a vertical direction into the water tank from the top to the bottom. The spinning speed was 50 m / min. The hollow fibers were then induced through a multistage water bath and a temperature increasing from 15 占 폚 to 40 占 폚. The wet hollow fibers discharged from the washing bath were dried in a convection type on-line drying step. After the texturing step, the hollow fibers were collected on a bundle shaped spinning wheel. Alternatively, a hand bundle could be formed.

Example  5

Hollow fiber Membrane  Produce

The polymer solution was prepared by dissolving distilled water as well as polyether sulfone (Ultrason TM 6020, BASF), polyvinylpyrrolidone (K30 and K85, BASF), polyurethane (Desmopan TM PU 9665 DU, Bayer MaterialScience AG) Methylpyrrolidone (NMP) at 50 占 폚 until a clear high-viscosity solution was obtained.

The weight fraction of the different components in the polymer spinning solution, PES / PVP (K85) / PVP (K30) / 9665 DU / H 2 O / NMP was 14/3/5/2/3/73. The viscosity of the resulting polymer solution was 22,900 mPa..

The warmed solution was cooled to 20 DEG C and degassed. Heating the polymer solution to 50 &lt; 0 &gt; C, and passing the solution through a spinning die. As a bore liquid, a water / NMP mixture containing 56% by weight of water and 44% by weight of NMP was used. The temperature of the die was 50 占 폚. The hollow fiber membrane was formed at a spinning speed of 40 m / min. The liquid capillaries exiting the die were passed into a water bath having ambient temperature. The distance between the die and the settling tank was 100 cm. The formed hollow fiber membrane was induced to pass through the water bath of the series. The wet hollow fiber membrane was then dried and had an internal diameter of 216 μm and an external diameter of 318 μm. The membrane had a fully asymmetric membrane structure. The active separation layer of the membrane was present inside. The active separation layer was defined as the layer having the smallest pore. The structure showed an overall sponge-like structure. The inner surface showed very smooth pores. The membrane was wound on a wedging wheel and a hand bundle with 200 fibers was prepared according to the method described below. The hydraulic permeability (Lp value) of the membrane was measured manually. The membrane exhibited a water permeability of 3.7 x 10 ^-4 cm ³ / (cm ² bar sec). Additionally, the constitution coefficient of myoglobin (in aqueous solution) was measured. After 15 minutes, a constitution factor of 1.5% was obtained, and at 60 minutes, a constitution factor of 1.1% was obtained. Then, the hydraulic permeability of the membrane (Lp value) was measured again, it was in ^{37 ℃ 2.7 x 10 -4 cm 3} / (cm 2 bar sec). Furthermore, experiments on diffusion transmittance of membranes were performed with chloride, inulin and vitamin B ₁₂ . Chloride, inulin and vitamin B ₁₂ for the transmittance were, respectively ^{10.5 x 10 -4 cm / sec,} 3.7 x 10 -4 cm / sec and 4.O x 10 ^-4 cm / sec at 37 ℃.

Example  6

Investigation of inserts and preparations for cell culture

The air-covered membranes (oxygen concentration was not increased or decreased) were treated with gamma irradiation (25 and 75 kGy, respectively) in a conventional sterile bag at room temperature for 6.3 and 18.9 h respectively. Water- or acrylic acid solution- The resulting membrane insert was treated with gamma irradiation in a plastic container containing approximately 70 ml of solution. To wash the fluid-covered membrane insert, approximately 300 ml of a conventional PBS medium (8 g NaCl, 0.2 g KCl, 1.44 g Na ₂ HPO ₄ , 0.24 g KH ₂ PO ₄ in 800 ml distilled water was dissolved and HCl , Adjusted to pH 7.4 using water, made up to 1 liter with water, and sterilized with autoclave) was prepared for each membrane type. The insert was sterilized from its container, washed in a PBS dish, and transferred to a 6-well plate for further rinsing. All insert types were transferred to 6-well plates and washed 5 times with 3 ml and 5 ml PBS on the top and bottom, respectively. For each of the cleaning steps, the inserts were incubated for at least 10 minutes to remove residual reagents and byproducts generated during gamma irradiation. After completing the cleaning procedure, the inserts were transferred to a new well plate for cell culture. In each well, 3 ml of MSC medium was added to the top, and 5 ml was added to the bottom of the insert. The inserts were incubated overnight in MSC medium in an incubator.

The fibronectin-coated membrane (based on the membrane according to Example 2), which is intended to be used for comparison, showed an exception. The insert was cleaned and cleaned as described above. At the top of the insert was added 1 ml PBS buffer containing 21 μg fibronectin and incubated overnight in the incubator. The next day, the fibronectin solution was removed, the insert was washed once with PBS, and incubated with cell culture medium before cell seeding. Untreated membrane inserts (Example 2) and TCPS culture plates were used as negative and positive control, respectively.

Example  7

Hollow fiber Membrane  Research

A bioreactor (with hollow fibers in the housing) treated with gamma irradiation was composed of the membrane of the present invention present as a hollow fiber geometry (see Examples 4 to 6). The materials for the housing, headers and potting were stable to gamma, and with Makrolon ^? (Fibasol blue (housing / header) and gamma stable polyurethane (potting) ^? DP1-1262 (Bayer MaterialScience AG). (RO-water), an aqueous solution of 0.001% acrylic acid (0.01 g AA in 1 l RO-water), or a 0.01% aqueous solution of PES / PVP / PA-based membrane (see Example 4) Filled with an aqueous solution of acrylic acid (0.1 g AA in 1 l RO-water) and treated with gamma irradiation at 25 or 75 kGy. Recharging of the reactor with aqueous solution and water was performed by flushing 100 ml / min from the IC (intra-capillary) side, and air was removed. The IC-out-line was fixed and the extra-capillary (EC) side was filled with ultrafiltration. The steps were repeated until no residual air remained in the bioreactor. Gamma irradiation was performed with a Co-source at 25 or 75 kGy applied for 6.3 and 18.9 hours at room temperature.

Example  8

flat bedsheet Culture of untreated bone marrow on membranes

MSC medium (900 ml alpha-MEM medium (Lonza, Cat. No. BE12-169F), 100 ml FBS, 10 ml penicillin / streptomycin and 10 ml ultra glutamine I; ) Was incubated overnight on the insert membrane and replaced with fresh MSC medium (2 ml) to the top of the insert (4 ml) and to the bottom (4 ml). These medium volumes were applied at every additional step. Each volume of untreated bone marrow was added to each insert as shown in Table I. The bone marrow was homogeneously distributed by vibrating the dish on the membrane. The inserts were placed in the incubator for 3 days to permit MSC adhesion. On day 3, the medium was removed from the top and bottom sides of the insert culture. Membranes were washed twice from the normal side with 2 ml PBS for each washing step. A new MSC medium was added to the top and bottom sides. In the first growing phase (i.e., the incubator from bone marrow seeding to the first MSC desorption), the medium was replaced on the top and bottom sides of the insert every 2-3 days until 12 or 14 days. On day 12 or 14, the MSC was desorbed from three of the four inserts using 500 μl trypsin per insert (10 min, 37 ° C). Trypsin was inactivated using 1.5 ml MSC medium per insert, MSC suspension was collected in a sterile tube, and samples were taken for MSC counts using a CASY counter. One of the four inserts was fixed and prepared for SEM. Therefore, the insert was washed once with PBS on the normal side, 1 ml of 2% glutaraldehyde solution was added, and stored at 40 ° C overnight. The insert was then washed three times with distilled water, air dried and treated with SEM. In the second experiment, no SEM was performed. The new medium was added to the top and bottom sides of the same insert after pre-warming and gas-conditioning of the MSC medium in the incubator until rescaling of the MSCs. 10,500 MSC / cm &lt; ² &gt; were reattached overnight on the same insert. In the second growth phase, reattached MSCs were grown on the membrane for an additional 7 days. MSC medium was changed every 2 to 3 days. MSCs were harvested from three inserts of four inserts at 19 or 21 days using trypsin for 10 min at 37 &lt; 0 &gt; C. The MSCs were re-plated on TCPS to contrast MSC morphology and proliferative potential. Therefore, the collected MSC to 5,000 MSC / cm ² for the control of the shape, and grinding was retry on a 4 or 5 days to TCPS 500 MSC / cm ² for the proliferation control for. Morphology and proliferative potential were recorded on day 1 or day 2 for morphological evaluation and on day 4 or 5 for microscopic evaluation of proliferative potential.

The untreated bone marrow was plated on various substrates ( Table I ). TCPS or VTK-FN (fibronectin-coated) and VTK (steam sterilized and uncoated) were used as positive and negative controls, respectively. After the first growth phase of 12-14 days, trypsinization was counted. The number of MSCs for TCPS is shown in pale color in FIGS. 1 and 2 . For the second growth phase, MSCs were re-plated onto the same insert at a density of 500 MSCs / cm ² and grown for a further 10 days into the second growth phase. After 7 days of the second growing period, measurements of MSC number (labeled black in FIGS. 1 and 2), re-plating for morphology (FIG. 3) and immobilization for SEM were performed. The number of MSCs for TCPS as a standard (n = 3) is shown in Figures 1 and 2 , and the lines showed standard TCPS levels. Experiments with untreated bone marrow were performed twice.

[Table I]

Overview of membrane types and irradiation conditions used to culture MSCs from untreated bone marrow

"n" represents the number of tests performed for each set-up. The expression "VTK" refers to the membrane based on the polymer solution used in Example 2. "FN" refers to "fibronectin". "The membrane AN69ST ™ is a polyacrylonitrile Based membrane.

As can be seen from Figures 1 to 3 , MSCs showed growth on fibronectin-coated VTK membranes in the first growth phase comparable to TCPS, i.e., fibronectin-coated VTK membranes are suitable for re-seeding cultures Did not do it. The reason for the lack of performance of the fibronectin-coated membrane after tripping is presumed to be degradation of fibronectin by trypsin. In experiment 2 ( FIG. 2 ), the fibronectin-coated VTK membrane did not act in either the first growth or the second growth. Uncoated VTK membranes showed very weak growth of MSC in both experiments. The air-covered VTK inserts illuminated by 25 kGy showed the opposite result when comparing experiments 1 and 2: in Figure 1 , the membrane type showed comparable performance to TCPS in the first growth phase, Showed a strong decrease of MSC in the growth phase, whereas the membrane type behaved in the opposite direction from experiment 2. The air-covered VTK inserts irradiated by 75 kGy showed comparable or better growth to TCPS at both growth periods. The coverage of the VTK membrane by aqueous acrylic acid solution and water with different concentrations during the gamma irradiation procedure resulted in results similar to the air-covered VTK inserts examined by 75 kGy. The AN69ST insert was included in Experiment 1 and proved to be unsuitable for MSC culture. After the second growth phase, the re-plated MSC showed mostly spindle-shaped morphology and normal growth (see FIG. 3 ).

Example  9

flat bedsheet Membrane  Lt; / RTI &gt; MSC Cultivation of

The MSC medium was incubated overnight and then replaced with fresh medium at the top and bottom of the insert from the insert. The MSC was applied to standard MSC flask conditions (0.25% trypsin, 5 min, and 37 ° C, knocking until all cells were desorbed and inactivation of trypsin by addition of medium) Respectively. The MSC was counted using a CASY counter. The MSC suspension was prepared by seeding 500 MSCs / cm &lt; ² &gt; in TCPS-wells or inserts. The well-plate and insert were placed in an incubator to permit MSC adhesion. MSC medium was targeted at confluence of -80% or less in TCPS until 7 or 9 days) were replaced at the normal and bottom sides every 2-3 days. On days 7 and / or 9, the MSCs were desorbed using 500 [mu] l trypsin (10 min, 37 [deg.] C). The trypsin was then inactivated using 1.5 ml MSC medium and MSCs were collected as a cell suspension in a sterile tube. Samples were taken for cell counting using a CASY counter. One insert was immobilized and prepared for SEM. Therefore, the insert was washed once with 1 ml of PBS on the normal side, followed by 1 ml of 2% glutaraldehyde solution, and all at 4 ° C overnight. Here, the insert was washed with distilled water, air dried and treated with SEM. New MSC medium was added to the top (2 ml) and bottom (4 ml) of the insert. The medium was prewarmed until cell re-seeding and conditioned with gas. 500 MSC / cm &lt; ² &gt; were reattached on the same insert in an incubator. Re-attached MSCs were propagated on the membrane for an additional 10 or 11 days. MSC medium was changed every 2 to 3 days. MSCs were collected from three inserts on days 16 and 21. MS was re-plated on TCPS for comparison of MSC morphology and proliferative potential. The MSCs were re-plated on TCPS for comparison of MSC morphology and proliferative potential. Therefore, the collected MSCs were re-seeded onto TCPS at 5,000 MSC / cm ² for control of form and 500 MSC / cm ² for control of proliferation for 4 or 5 days. Morphology and proliferative potential were recorded on day 1 or 2 for form evaluation and on day 4 or 5 for microscopic evaluation of proliferative potential. Table II provides an overview of all the membrane types and strains used in these experiments as well as the irradiation conditions.

[Table II]

An overview of the membrane type and irradiation conditions used to culture pre-selected MSCs

"n" represents the number of tests performed for each setup. "AA" represents "acrylic acid ". The expression "VTK" represents a membrane based on the polymer solution used in Example 2. [ "FN" refers to "fibronectin".

Experiments using preselected MSCs were performed twice and results are shown in FIG. 4 (Experiment 1) and FIG. 5 (Experiment 2). MSC showed growth on fibronectin-coated VTK membranes in the first growth phase of Experiments 1 and 2 comparable to TCPS, but showed strongly reduced growth in the second growth phase, i.e., the fibronectin- - Not suitable for seeding. The reason for the lack of performance of the fibronectin-coated membrane after trypticin was presumed to be degradation of fibronectin by trypsin. Uncoated VTK membranes showed very weak growth of MSC in both experiments. Air-covered VTK inserts irradiated by 25 kGy were tested only for Experiment 2, comparable to TCPS and comparable to all VTK membrane types after 75 kGy gamma irradiation. The air-covered VTK inserts investigated by 75 kGy showed MSC growth in the positive growth phase which is comparable to TCPS or better than TCPS in most cases. The coverage of the VTK membrane by aqueous acrylic acid solution and water with different concentrations during the gamma irradiation procedure resulted in results similar to the air-covered VTK inserts irradiated by 75 kGy. The AN69ST insert was included in Experiment 2 and proved unsuitable for MSC culture. The re-plated MSC on a conventional TCPS dish after the second growing period showed mostly spindle shaped morphology and normal growth.

Example  11

Steam before gamma irradiation Sterilized  effect

Experiments in accordance with Example 10 were performed, wherein the number of MSCs adhered to the membrane surface relative to TCPS was measured (%). Cell numbers were measured by air or water-filled VTK membranes irradiated by 25 or 75 kGy as well as TCPS and VTK membranes with fibronectin coating. Three sets of experiments were performed. In the first set, the membrane was irradiated directly after gamma irradiation. In the second set, the membranes were steam-sterilized prior to performing each gamma ray treatment. As can be seen from FIG. 6 , the steam-sterilized membrane prior to gamma irradiation performed better than the non-steam-sterilized membrane with respect to MSC attachment.

In the third set, the effect of additional EtO sterilization treatment steps was studied. The number of MSCs grown from the untreated bone marrow on the membrane irradiated by 75 kGy and w / o steam and EtO sterilized was significantly higher than that of MSCs grown on standard TCPS after the first (11 days) growing and the second (7 days) Of the total number of samples. As can be seen from Figure 7 , the EtO sterilization treatment did not have significant side effects on the performance of the membrane.

Example  12

Cell differentiation

In order to evaluate the differentiation ability of the cells cultured on the membrane according to the present invention, adipogenesis and osteogenesis differentiation tests were performed to measure the ability of MSCs to differentiate.

(A) Fatty cell formation

MSCs were seeded into 24-well plates at a density of approximately 10,000 MSC / cm &lt; ^{2 &} gt ;. Three wells, one well to differentiate MSCs into adipocytes as described below, one well to grow MSCs in standard MSC growth medium (without inducing adipocyte formation), and one well as a control for staining Were used. MSCs were grown in standard MSC growth medium (900 ml alpha-MEM medium, 100 ml FBS, 10 ml penicillin / streptomycin, and 10 ml ultra glutamine I; MSC medium was warmed to 37 ° C prior to cell culture use) Confluency or confluence for up to 10 days. Adipocyte formation was initiated in adipocyte inducing medium (20 μl of IBMX stock solution in 1 ml of standard MSC medium (55.55 mg of IBMX dissolved in 10 ml of distilled water, followed by 20 to 40 mg of sodium carbonate added to the solution), dexamethasone stock 1 μl of the solution (19.62 mg of dexamethasone in 50 ml of pure ethanol), 1 μl of insulin stock solution (10 mg / ml) and 2 μl of indomethacin (178.9 mg of indomethacin in 10 ml of pure ethanol) (0 to 11 days). The medium was changed every 2-3 days. Adipocyte-forming preservation medium (1 μl of insulin stock solution in 1 ml of standard MSC medium) was used for 3 or 5 days (12-14 days or 12-16 days) and replaced every 2-3 days. Cells were washed with standard PBS buffer. The cells were immobilized using formaldehyde solution (~ 10%) for more than 4 hours at room temperature. Subsequently, about 0.5 ml of an oil red O solution (fresh and sterilized and filtered) was added to each well, followed by incubation at room temperature for 30 minutes to 2 hours, the cells were washed twice with PBS, then 50% ethanol . Cells were counterstained with Mayer hematoxylin solution for 5 min at room temperature, washed three times for 1 min with tap water, and fixed with formaldehyde solution. Figure 8 shows the successful adipocyte formation of MSCs on cells cultured on VTK membranes in the presence of gamma irradiation (75 or 25 kGy, in the presence of acrylic acid or allylamine).

(B) Osteogenesis

The MSCs were seeded into 24-well plates at a density of approximately 10,000 MSC / cm. One well, one well for growing MSCs in standard MSC proliferation medium (without inducing bone formation), one well for dividing MSCs into osteoblasts as described below, one well as a control for staining Respectively. MSCs were grown to confluence or confluence using standard MSC medium or to 10 days. MSC medium is prepared by mixing 100 μl of glycerol phosphate stock solution (2.16 g of glycerol phosphate in 100 ml of standard MSC medium), 1 μl of dexamethasone stock solution and 1 μl of ascorbic acid stock solution (basal medium, 0.145 g of ascorbic acid in 10 ml of alpha-MEM)) and changed every 3-4 days. After 2-3 weeks, calcified sediments were present in or around the cells. Cells were washed in PBS. PBS was removed and the cells were fixed with 10% formaldehyde for 10 minutes, followed by one wash with PBS and two washes with deionized water. The cells were air dried and stained with silver nitrate for 10 minutes under UV light. After 2-3 washes with deionized water, the cells were counterstained with Mayer hematoxylin, then washed with tap water for 1 minute, and then washed twice with deionized water. Cells were embedded in 10% formaldehyde. Figure 9 shows successful bone formation of cells grown on membranes according to the present invention.

Example 13

Suitability for proliferation of various cell types

To evaluate the applicability of the gamma-irradiated membranes according to the present invention for culturing various cell types, flat membranes were tested by way of example. The membranes used were as described in Examples 10 and 11 and were referred to as "VTK" membranes. The membrane was examined with 25 kGy (air-covered) and 75 kGy (water-covered), respectively. Membranes were tested in short-term cultures, thereby assessing cell adhesion (1 day after seeding) and cell proliferation (5 days after re-seeding) compared to standard TCPS. The cells used were (a) NHDF, normal human dermal fibroblast, (b) HepG2, human liver cancer cell line, (c) HK-2, human kidney proximal epithelial cell line, and (d) MDCK, Madin- . Cells (a) to (d) were tested for membranes and TCPS according to the present invention. Cells were seeded as follows: (a) NHDF: 5 × 10 ³ cells / cm ^{2 in} medium (DMEM + 10% FBS + 1% penicillin / streptomycin); (b) HepG2: 5.6 x 10 ⁴ cells / cm ^{2 in} medium (RPMI + 10% FBS + 0.5% gentamicin); (c) HK-2: 10 in a medium (Keratinocyte SFM (Gibco, Cat # 17005) + Supplements for Keratinocyte SFM (Gibco, Cat # 37000-015) + 10 μg / ml Meronem + 1% FBS + 1% CaCl ₂ ) ⁴ cells / cm ² ; (d) MDCK: 10 ⁴ cells / cm ^{2 in} M199 + 10 μg / ml Meronem + 10% FBS (heat inactivated). Cell numbers were determined by CASY coefficients after cell seeding for 1 day days (proliferation) were measured. Figs. 10 to 13 shows that it could be of any cell type efficiently proliferate on 75 kGy irradiated membranes. the result is that the membrane was as efficient as at least a standard TCPS with respect to particular cell proliferation The results demonstrate that membranes treated with higher doses are somewhat better suited for cell culture than membranes treated with lower doses. The membrane VTK 25 kGy is effective for HK-2 cells ( Fig. 12) , presumably due to a problem in the first elimination of cells after the first day.

Example  14

Gamma-irradiated hollow fiber from untreated bone marrow Membrane (Bioreactor) MSC proliferation

Proliferation of MSCs from untreated bone marrow and proliferation of preselected MSCs were performed in a variety of hollow fiber bioreactors at the Cell Expansion System (CES). A bioreactor that has been proven to be suitable for pre-selected MSCs was further tested with untreated bone marrow. In the bioreactor according to the present invention, the doubling time was compared with the doubling time in a control flask (TCPS) starting at the same cell density when seeding. Doubling time served as a way to evaluate the performance of various membranes / TCPS.

Preselected MSC : The MSC used was pre-selected cryopreserved MSC derived from bone marrow with maximal passages 4. MSCs were pre-selected by proliferation of bone marrow-derived MSCs in conventional TCPS flasks with two or more subcultures. Approximately 2.8 Mio MSCs were loaded into a 1.7 m ² bioreactor corresponding to 164 MS / cm ² . When the surface area is smaller than 1.7 m ² , the number of loaded MSCs is evaluated. The incubation period was 7 days.

Untreated bone marrow : Approximately 10-12 ml of bone marrow was loaded into a 1.7 m ² bioreactor and the volume of loaded bone marrow was assessed if the surface area was less than 1.7 m ² . The incubation period was 13 days.

The T75 TCPS flask served as the control flask. The pre-selected MSCs were seeded into the flask at the same density (164 MSC / cm ² ) as in the bioreactor. When the untreated bone marrow was the starting material, 234 μl of bone marrow was seeded into the flask (3.12 μl / cm ² ) And 12 ml of bone marrow were loaded into a 1.7 m ² bioreactor (0.71 μl / cm ² ).

The membrane types used in this experiment are summarized in Table III below. Membranes based on PES / PVP / PA were prepared as described in Example 3. Membranes based on PES / PVP / PU were prepared as described in Example 5. The expression PAN means polyacrylonitrile.

[Table III]

Overview of membrane types tested for proliferation of pre-selected MSCs and untreated bone marrow cells

The results for the membranes were compared with standard TCPS flasks. Membrane 1, which was not irradiated with gamma radiation but was instead coated with fibronectin, was included in this experiment for comparative reasons.

The bone marrow was seeded into the bioreactor and the flask on day 0. On day 2, the medium was changed in the first cycle and then again on days 4 or 5, on days 6 or 7, on days 8-10, and on days 10-12. On day 13, cells were harvested (trypsin) and counted. The viability of the cells as well as their phenotype and morphology and / or proliferative behavior were examined.

Untreated bone marrow ( CFC Cells for calculation of cell number when using -F) Colony  Coefficient

78 [mu] l of untreated bone marrow were seeded into T25 (TCPS) flasks containing 5 ml MSC medium. Non-adherent cells were washed 2 to 3 days later by washing with PBS, and then fresh MSC medium was added. MSC medium was changed every 2 to 3 days. On day 7, colonies were counted (10x magnification / microscope).

Doubling time and doubling time ratio calculation

Doubling time (DT) is the period of time required for the cell to multiply by number. The DT and DT ratios of the MSC were calculated as follows.

MSC times the number of times = log ₂ (N / N ₀ )

N is the SUI of the MSC observed after the indicated incubation times, N ₀ is the number of seeded MSC. DT (time) = incubation time period (h) / doubling times. DT ratio = DT of cells in the bioreactor / DT of cells in the control flask

All selected gamma irradiation bioreactors with untreated bone marrow showed comparable performance to TCPS and VTK-FN bioreactors. Doubling times ranged from 28 to 34 hours, which was only slightly higher than the control flask (23 to 25 hours). Therefore, the doubling time ratio ranged from 1.1 to 1.5 ( Fig. 14) . The 0.5% PU bioreactor showed weaker slower cell growth compared to the gamma-irradiated bioreactor and the VTK-FN bioreactor.

The viability of the collected cells ( Figure 15 ) was tested by FACS analysis by 50,000 cells per FACS tube in MSC medium. One sample was stained with propidium iodide solution (target concentration 1 [mu] g / ml) in the dark at room temperature for 10 to 15 minutes. Another non-stained sample was used as a reference. The viability of the collected cells ranged from 88 to 99.5%.

The phenotype of the collected cells (see FIG. 16 ) was tested after trip myth of the cells. One million cells were resuspended in 5,000 [mu] l of blocking buffer (cell wash + 10% human serum) and incubated at 40 [deg.] C for 30 minutes. 2 μl of CD34-PE, 2 μl of CD45-PE, 2 μl of CD73-PE, mlgGl-PE - 2 μl of CD34-PE, and 50 μl of each were distributed into 10 tubes and the antibody was added FITC-1 μl (isotype control for HLA-DR), CD90-FITC-1 (1 μl isotype control for CD34, CD45 and CD73), HLA-DR, DP, DQ-FITC-2 μl and mIgG2a- The mixture was mixed well and stored in the dark at room temperature for 20 minutes. Then, FACS buffer (cells (1 ml), CD105-FITC (1 μl), mlgGl-FITC-2 μl (CD90, isotype control for CD105) Wash buffer + 2% FBS (heat-inactivated) was added, vortexed, and then centrifuged for 3 minutes at 400 g. The buffer was removed and the pellet was vortexed. 400 [mu] l was added and vortexed. Cells were stored at 40 &lt; 0 &gt; C and analyzed within 4 days.

Gamma-transformed, water-filled and cells proliferated in the bioreactor irradiated by 75 kGy showed relatively high values for CD45 and HLA-DR (18% and 16.7% of cells are positive, respectively). Cells collected from all other bioreactors exhibited the expected phenotype of MSC.

Examination of reattachment on TCPS after proliferation showed most spindle-shaped cells showing normal growth behavior after re-plating (data not shown).

Example 15

Influence of radiation dose

To investigate the effect of radiation dose on membrane characteristics, Example 14 was repeated with pre-selected MSCs using air-filled VTK membranes irradiated by gamma radiation doses of 12.5 kGy to 125 kGy, The number of grown MSCs relative to the number of MSCs grown on standard TCPS after the first and second (11 days) growth phase were determined. The results are shown in Fig.

Claims (17)

A membrane for cell culture comprising a hydrophilic polymer selected from the group consisting of polysulfone, polyethersulfone and polyarylether sulfone, and polyvinylpyrrolidone,
The membrane is covered by air and irradiated by gamma or beta radiation or electron beam at a dose of 70 kGy to 175 kGy in the presence of 4 to 100 volume% oxygen. The membrane of claim 1, further comprising a polyurethane. 3. Membrane according to claim 1 or 2, further comprising a polyamide. 3. Membrane according to claim 1 or 2, wherein the membrane is a hollow fiber membrane. 3. Membrane according to claim 1 or 2, wherein the membrane is a flat sheet membrane. 10. A method of manufacturing the membrane of claim 1 or 2,
(a) covering the preformed membrane with air,
(b) irradiating the membrane with a gamma ray, a beta ray or an electron beam at a dose of 70 kGy to 175 kGy
&Lt; / RTI &gt; A cell culture apparatus comprising the membrane of claim 1. Cell, and a membrane according to claim 1. An apparatus for in vitro treatment of body fluids, comprising: A method for culturing cells using the membrane of claim 1. 10. The method of claim 9, wherein the cells are adherent cells. 11. The method of claim 10, wherein the adherent cells are mesenchymal stem cells. delete delete delete delete delete delete

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